Human pluripotent stem cells, which include both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), hold the potential to differentiate into any cell type. As such, they can serve as comprehensive model systems of human cell genesis, particularly at early developmental stages that would otherwise be inaccessible to investigation. In addition, patient-derived hiPSC lines have a unique capacity to model human disease, although the scope of disorders amenable to this form of study is limited. Major considerations when creating hiPSC disease models include the capacity to efficiently generate, identify and isolate relevant cell populations, as well as recapitulate and assay critical aspects of the disease mechanism.
Retinal cell types are particularly well-suited for the investigation of cell development and dysfunction using pluripotent stem cell technology. The vertebrate retina harbors a modest repertoire of major cell classes sequentially produced via a conserved series of events. Furthermore, the effects of inherited and acquired retinal degenerative diseases (RDD) are often limited initially to a specific cell class, which simplifies the study of cellular mechanisms that incite RDD and the evaluation of potential therapies.
Previous studies have demonstrated the ability of human pluripotent stem cells to differentiate along the retinal lineage with varying efficiencies, with one protocol achieving a near uniform retinal cell fate using the WA01 hESC line (Lamba et al., 2011). However, pluripotent stem cell-derived retinal cells, particularly those from hiPSCs, are most often found in mixed populations that include some non-retinal or unidentified cell types. Further complicating matters is the fact that several markers used for retinal cell identification (e.g., calretinin, PKCα, Tuj1) also label cells found in other regions of the CNS. As such, a means to isolate developmentally synchronized populations of multipotent retinal progenitor cells (RPCs) across multiple hESC and hiPSC lines would be desirable. The RPCs and their definitive retinal progeny could then be used to study mechanisms of human retinal development and disease, examine retinal cell function, and devise and test RDD treatments.
In recent years, several groups have described the ability to direct human pluripotent stem cells (hPSCs) to a retinal fate (Lamba et al., 2006, 2010; Osakada et al., 2008; Carr et al., 2009; Hirami et al., 2009; Nakano et al., 2012; Buchholz et al., 2013). In order to serve as an effective in vitro model for human retinogenesis, as well as provide a foundation for translational applications, the stepwise differentiation of hPSCs through all of the major stages of retinogenesis helps to ensure the proper differentiation and prospective identification of hPSC-derived retinal progeny (Meyer et al., 2009, 2011; Gamm and Meyer, 2010; Sridhar et al., 2013).
The present inventors have previously described a method to differentiate human pluripotent stem cells to RPCs, retinal pigment epithelium (RPE), and photoreceptor-like cells in a manner that mimicked normal human retinogenesis (Meyer et al., 2009; the entire contents of which are incorporated herein by reference). However, a means to separate and track the fate of the RPCs in live culture was not available at that time. The present inventors have also described a method wherein transient morphological features were used to isolate structures with characteristics reminiscent of the optic vesicle (OV) (Meyer et al., 2011; the entire contents of which are incorporated herein by reference). Using such OV-like structures, it was possible to study principles of early human retinal development, monitor the sequence and timing of neuroretinal cell genesis, and optimize RPC and RPE production efficiencies in recalcitrant hiPSC lines.